The primary objective of this project is to define molecular and biochemical mechanisms involved in eukaryotic DNA synthesis. To approach this problem, we are investigating adeno-associated virus (AAV) DNA replication in both in vivo and in vitro systems. Current results show that AAV DNA synthesis can be initiated in vitro and that replicating forms that correspond to those identified in vivo can be synthesized with either endogenous or exogenously added templates. Recently, we have identified two DNA-associated proteins that may play an important role (one or both proteins) in the required processing of self-primed unit length hairpin and concatemeric intermediates of DNA replication. The observed mode of AAV DNA replication may serve as a model for telomere replication in eukaryotes. Among methods used are affinity chromatography, gel electrophoresis, DNA sequence analysis and restriction cleavage of DNA molecules.